Lamin B1 and nuclear morphology in peripheral cells as new potential biomarkers to follow treatment response in Huntington's disease

Dear Editor In neurodegenerative diseases, neuronal dysfunction and degeneration start many years before the emergence of clinical symptoms. An important goal to advance in their knowledge and treatment is the identification of peripherical biomarkers to predict the onset and progression and to test the efficacy of therapies.1 We have previously shown that alterations in lamin B1, a member of the lamin family of proteins that are crucial for nuclear functionality,2 are involved in the pathophysiology of Huntington’s disease (HD). Specifically, lamin B1 levels are increased in the R6/1 HD mouse model at the onset of motor symptoms in striatal medium-sized spiny and CA1 hippocampal neurons nuclei in correlationwith nuclear dysfunction.3 Therefore, we asked if increased lamin B1 levels and/or alterations in nuclearmorphology could be also occurring inmore accessible cells, namely fibroblasts and blood cells, and serve as biomarkers of the disease progression and/or treatment efficacy. Here, we show that HD patients’ peripheral cells display increased lamin B1 levels and alterations in nuclear morphology in a CAG-dependent manner as early as at pre-symptomatic stages. In R6/1 mice, fibroblasts and peripheral blood mononuclear cells (PBMCs) show intact lamin B1 levels but altered nuclear morphology at different stages of the disease, changes that were prevented by betulinic acid (BA) administration. Our data suggest the analysis of laminB1 protein levels and nuclearmorphology in peripheral cells as possible biomarkers for monitoring pharmacological treatments in HD. Lamin B1 protein levels were analysed by Western blot in primary fibroblast cultures derived from non-affected individuals (control) and from HD patients (Table S1). HD patients’ fibroblasts displayed increased lamin B1 levels in comparison to control ones (Figure 1A)with no correlation with the stage of disease progression, the presence or not of depression, or age (Figure S1A–C). Hence, HD fibroblasts


Lamin B1 and nuclear morphology in peripheral cells as new potential biomarkers to follow treatment response in Huntington's disease
Dear Editor In neurodegenerative diseases, neuronal dysfunction and degeneration start many years before the emergence of clinical symptoms. An important goal to advance in their knowledge and treatment is the identification of peripherical biomarkers to predict the onset and progression and to test the efficacy of therapies. 1 We have previously shown that alterations in lamin B1, a member of the lamin family of proteins that are crucial for nuclear functionality, 2 are involved in the pathophysiology of Huntington's disease (HD). Specifically, lamin B1 levels are increased in the R6/1 HD mouse model at the onset of motor symptoms in striatal medium-sized spiny and CA1 hippocampal neurons nuclei in correlation with nuclear dysfunction. 3 Therefore, we asked if increased lamin B1 levels and/or alterations in nuclear morphology could be also occurring in more accessible cells, namely fibroblasts and blood cells, and serve as biomarkers of the disease progression and/or treatment efficacy.
Here, we show that HD patients' peripheral cells display increased lamin B1 levels and alterations in nuclear morphology in a CAG-dependent manner as early as at pre-symptomatic stages. In R6/1 mice, fibroblasts and peripheral blood mononuclear cells (PBMCs) show intact lamin B1 levels but altered nuclear morphology at different stages of the disease, changes that were prevented by betulinic acid (BA) administration. Our data suggest the analysis of lamin B1 protein levels and nuclear morphology in peripheral cells as possible biomarkers for monitoring pharmacological treatments in HD.
Lamin B1 protein levels were analysed by Western blot in primary fibroblast cultures derived from non-affected individuals (control) and from HD patients (Table S1). HD patients' fibroblasts displayed increased lamin B1 levels in comparison to control ones ( Figure 1A) with no correlation with the stage of disease progression, the presence or not of depression, or age ( Figure S1A were classified depending on the number of CAG repeats. The CAG-repeats threshold at 42 was stabilised by paring CAG repeat length (Y axis) with lamin B1 protein levels (X axis) for each individual in a scatter plot ( Figure 1B). A positive correlation between lamin B1 levels and the number of CAG repeats was observed ( Figure 1C). Accordingly, only fibroblasts expressing mHTT with ≥42 CAG repeats showed increased lamin B1 levels ( Figure 1D), which was detected from pre-symptomatic stages ( Figure 1E) in all patients independently of the presence of depression ( Figure 1F), an early HD symptom. 4 Moreover, fibroblasts from HD patients expressing mHTT with ≥42 CAG repeats showed an increase in the presence of nuclear blebs, protrusions that correlate with nuclear functional alterations 5,6 ( Figure 1G). Lamin B1 levels tended to be increased in those nuclei showing blebs in both control and HD fibroblasts ( Figure S1D), a sign of correlation between elevated lamin B1 levels and the presence of blebs. BA administration to R6/1 mice reduces lamin B1 levels in a brain region-dependent manner. 3 In-line with our previous results, lamin B1 levels were normalized in HD patients' fibroblasts treated with BA, whereas this treatment had no effect on control ones ( Figure S1E,F).
Lamin B1 protein levels were also analysed in PBMCs by FACSI (see Table S2). Only B lymphocytes from HD patients showed altered lamin B1 levels ( Figure 2A) with a significant increase in those expressing mHTT with ≥42 CAG repeats ( Figure 2B), whereas nuclear circularity was only affected in those with <42 CAG repeats. We did not observe differences in lamin B1 intensity levels between disease stages nor did we observe an effect on lamin B1 levels due to age ( Figure S2A,C). Moreover, lamin B1 increased levels were only detected in B lymphocytes from HD patients without depression ( Figure S2B).
Mouse models allow to obtain longitudinal data that permit to correlate changes in peripheral cells with alterations in brain neurons. Thus, we asked whether fibroblasts and  PBMCs from R6/1 mice also show lamin B1 alterations. Moreover, as BA administration normalizes lamin B1 levels in the R6/1 mouse brain and in HD patients' fibroblasts, we analysed if the effects of this treatment could be monitored peripherally. Wild-type and R6/1 mice were treated from 8 to 20 weeks of age and blood, and fibroblasts were collected at different time points ( Figure 3A). In comparison to vehicle-treated wild-type mice, the number of fibroblasts presenting nuclear blebs was increased from 12 to 20 weeks of age in fibroblasts from vehicle-treated R6/1 mice and that was prevented by BA administration (Figure 3B,C). However, lamin B1 levels were not altered at any of the ages analysed ( Figure S3A). Moreover, in 20week-old mice, lamin B1 levels were increased in those fibroblasts showing nuclear blebs ( Figure S3B), suggesting that increased lamin B1 levels are involved in the formation of blebs. For the analysis of lamin B1 levels and nuclear morphology in PBMCs, we performed immuno-histochemistry in blood films as not enough quantity of blood was obtained for FACSI. PBMCs from 16-to 20-weekold vehicle-treated R6/1 mice displayed altered nuclear circularity in comparison to vehicle-treated wild-type littermates ( Figure 4A), although lamin B1 intensity was not altered at any of the ages analysed ( Figure S4A). Interestingly, BA administration delayed the appearance of this phenotype until 20 weeks of age ( Figure 4A). Moreover, in PBMCs with altered nuclear circularity at 20 weeks of age, lamin B1 levels tended to be increased ( Figure S4B).
In conclusion, lamin B1 protein levels are increased in HD patients' fibroblasts and B lymphocytes in a CAG length-dependent manner, correlating with alterations in nuclear morphology. In the R6/1 mouse model, although lamin B1 levels are not altered, fibroblasts and PBMCs show altered nuclear morphology that is ameliorated by BA administration ( Figure S5). Interestingly, the detection of these changes in stages previous to the development of F I G U R E 3 Betulinic acid treatment prevents the appearance of nuclear blebs in fibroblasts from R6/1 mouse. (A) Timeline of the experimental procedure used to assess the peripheral effects of betulinic acid (BA) in wild-type (WT) and R6/1 mice. Blood and fibroblasts samples from vehicle-and BA-treated WT and R6/1 mice were obtained at the indicated week (w) of treatment. (B) The percentage of cells presenting nuclear blebs was analysed by immunohistochemistry in fibroblasts from vehicle-or BA-treated WT and R6/1 mice at different stages of the disease (w, weeks). Each point corresponds to the value of an individual sample. Bars represent the mean ± S.E.M. (C) Representative images at 16 weeks of age are shown (white arrowheads: blebs). Scale bar 10 μm. One-way ANOVA followed by Bonferroni's post hoc test. **p < .01, ***p < .001, ****p < .0001 symptoms, both in HD patients and R6/1 mice, could help to an earlier implementation of neuroprotective therapies. Overall, our results support the usefulness of lamin B1 and nuclear morphology analysis in HD patients' fibroblasts or B lymphocytes to peripherally monitoring the effectiveness of drug treatments. University of Barcelona, for their support and advice concerning Cytometry and confocal techniques, respectively. We would like to also thank all patients and non-affected individuals for participating in this study. Ministerio de Ciencia e Innovación (MCIN), Agencia Estatal de Investigación (AIE/10.13039/501100011033), Spain (SAF2016-08573-R and PID2019-106447RB-I00 to EP-N, and PID2020-116474RB-I00 to VB), FIS PI21/01758, Fondos Feder 'una manera de hacer Europa', ISCIII, Spain to JK and Fundación Ramón Areces, Spain to EP-N; MG-F was supported by a grant (FI-2016) from Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) and CC-P by a grant from the University of Barcelona.